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Biological Effects of Ionizing Radiation: A Systems Biology Perspective

Biological Effects of Ionizing Radiation: A Systems Biology Perspective. Mary Helen Barcellos-Hoff New York University Departments of Radiation Oncology and Cell Biology School of Medicine. IRPA Glasgow May 14, 2012.

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Biological Effects of Ionizing Radiation: A Systems Biology Perspective

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  1. Biological Effects of Ionizing Radiation: A Systems Biology Perspective Mary Helen Barcellos-Hoff New York University Departments of Radiation Oncology and Cell Biology School of Medicine IRPA Glasgow May 14, 2012

  2. “Radiation is extraordinarily effective in eliciting biological effects” Dudley Goodhead, ICRR 1 Gy=1 J/kg100,000 ionizations/cell1000 SSB30 DSB20% clonogenic death

  3. “Radiation is a weak carcinogen” Barcellos-Hoff, ICRR 1 Gy~6% ERR1 cancer /100 people~1015 cells~1017 ionizations BEIR VII

  4. Why? Some irradiated tissues develop cancer. Radiation induces DNA damage and molecular responses in cells. Tissue respond to such signals.

  5. Radiation health effects are system, rather than component, driven. That is, a property of the tissue rather than the cell.

  6. What is Systems Biology?

  7. What is Systems Biology? • Systems biology addresses problems that cross scales of organization and time. • Uses quantitative measurements of the behavior of groups of interacting components, systematic measurement technologies such as genomics, bioinformatics and proteomics, and mathematical and computational models to describe and predict dynamical behavior. • Focus on dynamics is the main conceptual difference between systems biology and bioinformatics.

  8. Systems Biology Systems Biology Links Information Across Scales of Complexity and Time Physiology Cell Biology Molecular Biology Systems Biology

  9. Radiation Biology Radiation Biology is Systems Biology Reoxygenation Repopulation Repair Radiation Biology

  10. Systems Biology What distinguishes a complexsystem from a merely complicated one is that some behaviors emerge as a result of altered relationships between the elements. http://www.biologydaily.com/biology/Systems_thinking

  11. Phenotype Targeted DNA Damage Response Tissues Mutation Survival Intercellular Signaling Non-Targeted

  12. Targeted vs Non-Targeted Radiation Effects • Targeted effects are proportional to radiation dose, e.g. cell kill • Persistence through genetic sequence like mutation • Non-targeted effects are responses to damage and often have little dose dependence: switches rather rheostats • Persistence by altered interactions or phenotype

  13. Recognition of DNA Damage • Radiation-induced foci are now widely used to follow the rapid response to DNA damage • Double strand breaks (DSB) recruit MRN complex and activate kinase, ATM • ATM leads to phosphorylation of histone H2AX (gH2AX) • gH2AX relax chromatin structure for assembly of repair complex

  14. While many report proportionality between radiation exposure and response, they are not the same. Physical damage is nearly instant Response is biological, max 30 min Resolution is enzymatic and quite slow Other biological processes (e.g. DNA replication) also induce foci Clear discrepancies between predicted DNA DSB and detection of foci RIF are constrained by chromatin domains Dense chromatin do not exhibit foci at early time points even though damage is there. Biological modifiers can block response. Radiation-Induced Foci are Response, NOT Damage Costes et al, PLoS Computational Biology, 2007 Nudiemeir et al. PNAS, 2012

  15. Targeted Radiation Effects Alter Intracellular Signaling, Survival and Sequence • DNA damage recognition and repair • Complex but well-orchestrated series of protein modifications and recruitment to DNA strands that execute repair Signaling • Acute and delayed processes affect cell survival • Apoptosis • Autophagy • Necrosis • Mitotic catastrophe • Interphase death • Defective recognition or repair processes lead to mutations • Clonal sequence changes evident in surviving daughter cells Survival Sequence

  16. Properties of Cancer Cells • Self-sufficiency for growth • Disregard for anti-growth signals and programs • Limitless proliferative potential • Mutations and/or genomic instability • Aberrant nuclear architecture

  17. Properties of Cancers • Disrupted morphogenesis • Subversion of the host • Stimulate angiogenesis • Escape immune surveillance • Provoke inflammation • Recruit activated host cells, e.g. macrophages • Invasion and Metastasis

  18. Phenotype Non-Targeted Radiation Effects Alter Intercellular Signaling, Phenotype and Cell Interactions Tissues Signaling

  19. Sham IR TGFβ IR+TGFβ • Irradiated human epithelial cells treated with TGFβ undergo epithelial to mesenchymal transition (EMT). • Stem/progenitor cells increase. • The dose and radiation quality response switch like for both.

  20. Experimental Strategies for Identifying NTE and their Physiological Relevance Barellos-Hoff Health Physics, 2010

  21. Biological Challenges for NTE Roles in Cancer Risk • Are NTE operational in vivo? • Are NTE relevant to disease causation or modulation? • What is the rationale for including NTE in radiation protection and risk modeling?

  22. Oncogenic bystander radiation effects in Patched heterozygous mouse cerebellum Mariateresa Mancuso*, Emanuela Pasquali†, Simona Leonardi*, Mirella Tanori*, Simonetta Rebessi*, Vincenzo Di Majo*, Simonetta Pazzaglia*, Maria Pia Toni‡, Maria Pimpinella‡, Vincenzo Covelli*, and Anna Saran*§ PNAS 106:12445, 2008

  23. Protection via Cell-Interactions:Selective Apoptosis of Aberrant Cells • Low doses suppress in vitro transformation (Redpath, 2008) • Transformed cells are selectively deleted by signaling from normal cells and low dose irradiation augments the efficacy of normal cells (Bauer, 1996; O’Neil, 2007) • Radiation-induced TGFb deletes genomically unstable cells in vitro and in vivo (Barcellos-Hoff, 2008) • Low dose radiation suppresses recombination in vivo (Sykes, 2008)

  24. 2 Senescent fibroblasts produce matrix metaloproteinases SF (batch 1) SF (batch 2) NF MMP-1 MMP-3 MMP-7 1 Radiation induces a senescent phenotype in fibroblasts MMP-9 MMP-10 3 Radiation induced senescent fibroblasts alter epithelial morphogenesis and growth MMP-12 GAPDH Zhi-Min Huang Can Res 2006

  25. Radiation Induced Genomic Instability Kadhim, et al. (1994). "Alpha-particle-induced chromosomal instability in human bone marrow cells." Lancet344: 987-988. Kadhim, et al. (1995). "Radiation-induced genomic instability: delayed cytogenetic aberrations and apoptosis in primary human bone marrow cells." Int J Radiat Biol67: 287-293. Kadhim, et al. (1992). "Transmission of chromosomal instability after plutonium alpha-particle irradiation [see comments]." Nature355: 738-740. Lorimore, et al. (1998). "Chromosomal instability in the descendants of unirradiated surviving cells after alpha-particle irradiation." Proceedings of the National Academy of Sciences of the United States of America95: 5730-5733. Watson, et al. (1997). "Genetic factors influencing alpha-particle-induced chromosomal instability." International Journal of Radiation Biology71: 497-503. • Non-clonal chromosomal instability • Daughters of irradiated cells • Not dose dependent • Additional endpoints

  26. Radiation Induced Cell Interactions and Phenotypes Generate de novo DNA Damage Activated phagocytic cells produce genetic effects in co-culture MutationWeitzman & Stossel, Science, 212, 546, 1981 Cytogenetic changesWeitberg et.al., New. Eng.J.Med.,308, 26-30, 1983 Modification of DNA basesDizdaroglu et.al., Cancer.Res, 53,1269,1993 TransformationWeitzman et.al., Science, 237, 1231, 1985 Coates, et al. (2008). "Indirect macrophage responses to ionizing radiation: implications for genotype-dependent bystander signaling." Cancer Res68: 450-456. Gowans, et al. (2005). "Genotype-dependent induction of transmissible chromosomal instability by gamma-radiation and the benzene metabolite hydroquinone." Cancer Research65: 3527-3530. Lorimore, et al. (2008). "Chromosomal instability in unirradiated hemaopoietic cells induced by macrophages exposed in vivo to ionizing radiation." Cancer Res68: 8122-8126. Lorimore, et al. (2001). "Inflammatory-type responses after exposure to ionizing radiation in vivo: a mechanism for radiation-induced bystander effects?" Oncogene20: 7085-7095. Lorimore, S, et al. (2005). "Chromosomal Instability in Unirradiated Hemopoietic Cells Resulting from a Delayed In vivo Bystander Effect of {gamma} Radiation." Cancer Res65: 5668-5673. Radiation induces an “activated” macrophage phenotype Soluble signal generates DNA damage in neighbouring cell.

  27. Do non-targeted effects, i.e. radiation induced effects on intercellular signaling, phenotype and cell interactions, affect the development of cancer?

  28. Remove epithelium at 3 wk Irradiate at 3 mo Genetic/Radiation Mammary Chimera Transplant with oncogenic tissue.

  29. Irradiated Tissue Radiation Chimera Mice irradiated at 3 months. • Transplant with Trp53 fragment • Observe ~ 1 year Tissues collected across time for microarray, immunostaining, and stem cell analysis. Control Irradiated Host Evaluate tumor latency, histology, marker analysis and microarray. A Systems Radiation Biology Approach Comparison of tissue and tumor global expression profiles Identification of common and persistent pathways Validation and hypotheses testing

  30. What happens to the route to cancer in an irradiated mouse? • Aneuploidy, 8 months • Ductal carcinoma in situ, 8 months • Tumor latency, 12 months • Highly heterogeneous histology Initiating, rather than initiated, epithelium Jerry, D.J., et al. (2000). A mammary-specific model demonstrates the role of the p53 tumor suppressor gene in tumor development. Oncogene 19, 1052-1058.

  31. Host Irradiation Promotes Mammary Carcinogenesis Tumor latency of Trp53 null cancer is decreased by 63 days in irradiated mice. Tumor incidence at a year is increased 31% in irradiated hosts. The effect is not directly proportional to dose: 10 or 100 cGy host irradiation have a comparable effect. Once established, tumors grew faster in irradiated mice. p<0.05 Nguyen et al. Cancer Cell 2011

  32. Host Irradiation Affects the Tumor Estrogen Receptor Status • Estrogen receptor is the most important diagnostic feature of human breast cancer. ~2/3 are ER+. • In sham-irradiated hosts, most, 65% (28/45), Trp53 null cancers are ER+. • Only 35% (33/93) were ER+ in irradiated hosts. • Thus, host irradiation increased aggressive ER-negative tumors. Nguyen et al. Cancer Cell 2011

  33. ER Status of Radiation-Preceded Human Breast Cancer Castiglioni et al. Clinical Cancer Research 13:46, 2007 Radiation-preceded Sporadic tumors breast carcinomas (consecutive series) p Median age , y 39 57 0.0001 ER+, % 50 72.9 0.009 PgR+, % 31.2 58.3 0.002 p53+, % 35.8 10.2 0.001 Cytokeratin 5/6+, % 45.4 17.3 0.003 53% of breast cancers of women who were treated with radiation therapy between 14-20 years of age were ER-negative compared to 11% non-irradiated age matched patients (p< 0.0001).

  34. Radiation non-targeted effects affect cancer latency and features.

  35. What are the signals? Studying cancer as a system rather than a jigsaw puzzle • Global expression profiles of protein, DNA or mRNA are used to describe states • Bioinformatics can integrate multiple features in context and through time • Robust patterns emerge that define key cancer characteristics • Systematic analysis to identify ‘pressure’ points, i.e. hubs, that are key

  36. Gene Probes Differentially Regulated Across Samples ~2,000 All Gene Probes 30,000+ Gene Probes that Behave in a Similar Manner, i.e Gene Neighbors or Networks Genes That Discriminate Between Tumors Arising in Irradiated Hosts

  37. 8/9 Sham Gene Expression Profiles Discriminate Tumors Arising in Sham or Irradiated Hosts • 24 genes can cluster tumors from irradiated hosts vs sham-irradiated hosts. • Even though the irradiation occurred months before and in the absence of the cells giving rise to the tumors • Two key pathways, TGFβ and Notch, were identified and functionally validated Nguyen et al. submitted, 2010

  38. Radiation Does Not Promote Cancer When Host TGFβ is Compromised Neither latency of Trp53 null cancer nor incidence at a year is affected in irradiated Tgfb1 heterozygote mice. These data identify TGFβ as a key signal in the irradiated microenvironment. Suggest that blocking TGFβ activity after radiation could inhibit cancer. Nguyen et al. Cancer Cell 2011

  39. Processes described by these genes include: Innate immunity, i.e. macrophages Vascular integrity Inflammation Cytokine signaling Each contributes cancer in other experimental models Each is subject to therapeutic targeting to prevent or treat cancer. What processes occurring in tumors arising in an irradiated host are different? 323-IHC 156-IHC 24-IHC >1.5-Fold Change. 100% of SAMs. >2.0-Fold Change. 100% of SAMs. >1.5-Fold Change. 80% of SAMs.

  40. Gene Probes Differentially Regulated Across Samples ~2,000 All Gene Probes 30,000+ Gene Probes that Behave in a Similar Manner, i.e Gene Neighbors or Networks Genes That Discriminate Between Tumors Arising in Irradiated Hosts 24-IHC (2-fold) 156-IHC (1.5 fold) 323-IHC (1.25 fold) 323-IHC (1.25 fold) Mouse Genes Translated Into Human Genes

  41. Irradiated Host Signature Detects Radiation History in Human Cancer Radiotherapy associated sarcomas Radiation preceded thyroid cancers • 144 human genes from the 323-IHC clustered radiotherapy associated sarcomas (Hadj-Hamou, 2011). • 93 genes from the 323-IHC clustered radiation-preceded papillary thyroid carcinomas (Delys,, 2007). Cluster 3.0: Public data was pre-centered. Clustered 93 genes by uncentered correlation; arrays by spearman rank correlation; complete linkage.

  42. Challenges for Incorporating NTE into Risk Assessment • Are NTE operational in vivo? YES • Are NTE relevant to disease causation or modulation? YES • What is the utility for including NTE in radiation protection and risk modeling?

  43. Predicting radiation health effects requires understanding the relationship between targeted and non-targeted radiation effects. Non-targeted effects are avenues for protection after exposure. A biological model of cancer risk following radiation should incorporate systems biology principles of complexity and emergence. Non-Targeted Effects are Critical Mechanisms in Radiation Carcinogenesis

  44. What can be achieved by identifying multicellular processes in radiation carcinogenesis? • More complete understanding of radiation carcinogenesis feeds into biologically based cancer risk models for as yet unknown exposures, e.g. space travel • Tests the biological veracity of LNT for low dose/dose rate where epidemiological data are uncertain • Serves as a basis for assessing adequacy of protection standards • Used to identify susceptible populations • Could provide avenues for protection after radiation exposure

  45. U.S. Department of Energy Low Dose Program NASA Specialized Center of Research NIEHS Breast Cancer and the Environment

  46. Learning Objectives • Cancer biology has moved from cataloging aberrations in cancer cells to trying to understand the evolution of a tumor as a tissue • Non-targeted effects (NTE) of radiation contribute to carcinogenesis • Incorporating NTE into radiation risks is important as it offers routes for prevention and different consideration of dose dependence.

  47. Cancer is two-compartment problem in which mutant cells must override effective suppression by normal tissue. Radiation can increase carcinogenesis by acting on both compartments. While targeted effects like mutations are dose dependent, non-targeted effects act more like switches, which needs to be understood. Many biological models of radiation carcinogenesis are incomplete

  48. Luminal A HER2Normal Like Luminal B Basal-likeNo subtype Group 1 Group 2 Group 1A Group 1B Group 2C Group 2D Clustering of Human Breast Cancers by Irradiated Host Signature Unsupervised hierarchical clustering using 182 of the 323-IHC genes of 1,600 human breast cancers compiled by Staaf et al hat used the Affymetrix Human U133A platform. David Nguyen, Erik Freidlund and J. H. Mao

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